Method for preserving and stabilising proteins, which can be used for industrial development of formulations of sanitary, pharmaceutical and cosmetic products

ABSTRACT

A method for preserving and stabilising proteins is disclosed which can be used for the industrial development of formulations of sanitary, pharmaceutical and cosmetic products, particularly cell growth factors and/or proteins such as epidermal growth factor (EGF) and fibroblast growth factor (bFGF). The method includes a dispersion phase, under normal pressure and temperature conditions, in which the proteins are incorporated into an anhydrous medium formed by oily components that have hydrophilic residues and that guarantee interactions with the proteins, while maintaining the native conformation of the proteins, such components being grape seed oil, a base of different components and butylhydroxytoluene.

OBJECT OF THE INVENTION

The invention, as stated the title of the present specification, relatesto a method for preserving and stabilising proteins, which can be usedfor the industrial development of formulations of sanitary,pharmaceutical and cosmetic products.

The object of the invention is focused on a method which envisages thecreation of a dispersed system as a means for preservation,stabilization and storage of proteins in an oily phase under normalpressure and temperature conditions. Particularly, the method proposedby the invention is based on the incorporation of proteins, such as cellgrowth factors such as tales epidermal growth factor (EGF) andfibroblast growth factor (bFGF), although not limited thereto, in amedium composed of components, such as grape seed oil, a base consistingof various compounds that will be specified later in the presentdescription, and butylhydroxytoluene (BHT), that form the cited oilyphase. In particular, the method proposed takes advantage of somechemical groups of the oily phase components promote certainphysical-chemical interactions with the residues of proteins thatenhance for longer periods of time the maintenance of the nativemolecular structure of proteins, achieving that such method is simple,economical and of general application, with potential capacity toreplace complex preservation techniques and/or aqueous means commonlyused for the preservation of proteins.

FIELD OF THE INVENTION

The field of application of the present invention falls within theindustrial chemical sector, focusing particularly on the scope of theindustry dedicated to the manufacture of pharmaceutical and cosmeticproducts, in particular those intended to preserve growth factors.

BACKGROUND OF THE INVENTION

Currently the stability in proteins has attracted considerable interestin the chemical, pharmaceutical and cosmetic field. Proteins are used asactive substances of numerous treatments in diseases such as diabetes,cancer, haemophilia, myocardial infarction, just to cite somepathologies [Krishnamurthy and Manning, 2002]. Note that the use ofprotein structures such as growth factors, e.g. the EGF, aroused a greatinterest in the cosmetic industry in recent years. So, if a protein cannot be properly stabilized, it may lose its native structure withconsequent loss of its biological activity [Krishnamurthy and Manning,2002].

The problem is that the protein stabilization is particularly difficultsince they are very susceptible to degradation phenomena: physical,chemical and enzymatic. Chemical degradation is related to deamination,oxidation, reduction, hydrolysis processes and chemical interactionssuch as disulfide bond interactions. Physical degradation includessurface adsorption, aggregation, dissociation, denaturation, andphotolysis processes. In addition, there are factors that influence theaggregation of proteins related to the properties of the dispersionmedium, such as temperature (related to the thermodynamics and kineticsof transformation of the structural protein conformation), pH (relatedto interactions of positive or negative charges with the proteinresidues), ionic strength (related to salts and their concentration tointeract with charged groups) and surfactants (related to conformationalthermodynamic stability) [Chi et al., 2003].

There are technological processes to ensure that proteins remain forlonger period of time with its native conformation, said processes canbe carried out by physical processes such as freezing (below −10° C.) orlyophilisation (for the elimination of the humidity present in anaqueous solution of protein), however, even the products obtained bythese methods suffer from degradation; or chemical processes through theaddition of co-solvents may be carried out [Chang and Pikal, 2009].

The EGF was the first polypeptide isolated and characterized as a growthfactor. It has a biological activity related to its native structurecapable of stimulating the proliferation of keratinocytes andfibroblasts (with the consequent formation of collagen), inducesangiogenesis (formation of new vessels) and performs subsequentvascularisation of the area where it is applied. These propertiespromote the appearance of new skin with a considerable thick, restoringits elasticity and firmness, thus diminishing the unwanted effects ofcellular oxidation and therefore resulting in the elimination ofwrinkles [Tang et al., 1994]. Such growth factor has begun to userecently in topical formulations, where very good results have beenobtained related to tissue regeneration, the acceleration in the healingof burns, treatment of keloid, acne and stretch marks, even improvingoutcomes of treatments of surgical type, promotion of the consolidationof skin grafts as well as the post-peeling application. However, saidproteins are increasingly used in the pharmaceutical and cosmeticindustry, but have not been used massively due to its high prices andtheir difficult stabilisation [Schouest et al., 2012].

Fibroblast growth factor (bFGF) is a growth factor that acts to increasethe mitotic activity index and DNA synthesis, facilitating theproliferation of various precursor cells, such as chondroblasts,collagenoblasts, and osteoblasts, etc., that form the body's fibrous,connective, and support tissues. It contributes to wound healing,haematopoiesis, angiogenesis, or the embryonic development. To this end,they perform very different functions: a) they contribute to there-epithelialisation of the tissues damaged during healing; b) they haveblood vessel formation inducing-activity; c) they are involved inprocesses for differentiation of the blood cell lines; and, d) they areinvolved in the differentiation of skeletal and cardiac muscle, thematuration of the lungs and the specification of the hepatocytes fromendoderm cells.

The object of the present invention is, therefore, the development of anew method for preserving and stabilising proteins, which can be usedfor the industrial development of formulations of sanitary,pharmaceutical and cosmetic products that, unlike conventional methods,it takes place in oily phase so that it is more simple and economical,having noted that, at least by the applicant, the existence of anydocument or invention that discloses a method for preserving andstabilising proteins or similar invention that has technicalcharacteristics similar to those here proposed is unaware.

EXPLANATION OF THE INVENTION

A medium for dispersing proteins the components of which provides themedium with an oily character has been developed. Thus, the EGF, bFGFand other cell growth factors and/or proteins, as stated, aremacromolecules with difficult stabilization, since, currently, themajority of dispersion means have aqueous character and achieve thestabilization in a short period of time, through the use of dispersedsystems such as emulsions, or expensive and very specific methods thatdo not ensure a longer life of the protein native structure, such aslyophilisation.

With the present invention, the development of the EGF and other growthfactors and/or proteins has been carried out in oily medium as anintermediate product for use in specific industrial processes, promotingthe stability of proteins with respect to the currently existing methodsthus achieving a more effective action to the not denatured proteins insuch medium not being denatured the proteins in such medium.

More specifically, according to the method of the present invention, inthe formulation the growth factors are surrounded by an anhydrous mediumcomposed of other components that act as adjuvants interacting with theresidues of proteins, such components being: grape seed oil, thatcreates a medium which reduces the electrostatic interactions with theprotein residues while maintaining the native conformation of theproteins; base consisting of: Caprylic/Capric triglyceride; PEG-18castor oil dideate; Propylene glycol; Pentaerythrityl tetra Di-T-Benzylhydroxy hydrocinnamate; tocopherol, Trisisopropanolamine that promotesthe interactions by intermolecular forces with the domains of proteinsmaking the structure thermodynamically more stable; andbutylhydroxytoluene (BHT), which acts as an antioxidant, largelyavoiding phenomena of chemical degradation by oxidation in proteins.

Note that the inter-position process of protein with the oily phasecomponents is carried out with appropriate quality; such a process ispreferably carried out in clean rooms, under laminar flow conditions,where environmental quality controls as well as microbiological controlsare guaranteed to ensure the sterility of the product.

In short and succinctly, the present invention proposes the developmentof a method for the preservation, storage and stabilization of proteinswhich contemplates an anhydrous dispersion phase (i.e., in the absenceof water) with environmental and microbiological quality, through theapplication of oily substances having hydrophilic residues thatguarantee interactions with the proteins that keep its conformation inthe native state, constituting a reproducible, simple and economicmethod with regard to others methods that require the use of devices,complex methods and qualified personnel.

Having sufficiently described the nature of the present invention, aswell as a way of putting it into practice, it is not considerednecessary to make a more extensive explanation in order that any expertin this area will understand its scope and the advantages that can bederived from it, making known that, within reason it could be put intopractice in other embodiments differing in detail from that indicated byway of example, and which will obtain the same degree of protection,provided that they do not alter, change, or modify its fundamentalprinciple.

BIBLIOGRAPHY

-   Chang L L, Pikal M J. Mechanisms of Protein Stabilization in the    Solid State. J. Pharm. Sci. 98; 2009: 2886-2908.-   Chi E Y, Krishnan S, Randolph T W, Carpenter J F. Physical stability    of proteins in aqueous solution: mechanism and driving forces in    nonnative protein aggregation. Pharm Res. 2003; 20: 1325-36.-   Krishnamurthy R, Manning M C. The stability factor: importance in    formulation development. Curr Pharm Biotechnol. 2002; 3: 361-71.-   Schouest J M, Lun T K, Moy R L. Improved texture and appearance of    barley produced, synthetic, human-like epidermal growth factor (EGF)    serum. J. Drugs Dermatol. 2012; 11 (5): 613-620.

Tang Z, Zhang Z, Zheng Y et al. Cell aging of human diploid fibroblastsis associated with changes in responsiveness to epidermal growth factorand changes in HER-2 expression. Mechanisms of Ageing and Development1994; 73 (1): 57-67

1. Method for preserving and stabilising proteins, which can be used forthe industrial development of formulations of sanitary, pharmaceuticaland cosmetic products, particularly cell growth factors and/or proteins,such as the epidermal growth factor (EGF) and fibroblast growth factor(bFGF), characterised in that it comprises a dispersion phase, undernormal pressure and temperature conditions, in which the proteins areincorporated into an anhydrous medium formed by oily components havinghydrophilic residues and guarantee interactions with the proteins whilemaintaining the native conformation of the proteins.
 2. Method forpreserving and stabilising proteins, which can be used for theindustrial development of formulations of sanitary, pharmaceutical andcosmetic products, according to claim 1, characterized in that the oilyphase components comprise grape seed oil.
 3. Method for preserving andstabilising proteins, which can be used for the industrial developmentof formulations of sanitary, pharmaceutical and cosmetic products,according to claim 1, characterised in that the oily phase componentscomprise abase consisting of Caprylic/Capric triglyceride; PEG-18 castoroil dideate; Propylene glycol; Pentaerythrityl tetra Di-T-Benzyl hydroxyhydrocinnamate; tocopherol, Trisisopropanolamine.
 4. Method forpreserving and stabilising proteins, which can be used for theindustrial development of formulations of sanitary, pharmaceutical andcosmetic products, according to claim 1, characterised in that the oilyphase components comprise butylhydroxytoluene.
 5. Method for preservingand stabilising proteins, which can be used for the industrialdevelopment of formulations of sanitary, pharmaceutical and cosmeticproducts, according to claim 1, characterised in that the oily phasecomponents are grape seed oil, butylhydroxytoluene and a base consistingof: Caprylic/Capric triglyceride; PEG-18 castor oil dideate; Propyleneglycol; Pentaerythrityl tetra Di-T-Benzyl hydroxy hydrocinnamate;tocopherol, Trisisopropanolamine.